A very large number of diterpenoids possessing a labdane skeleton (FIG. 1) 
occur in nature (Connoly, J. D.; Hill, R. A Dictionary of Terpenoids, Chapman and Hall: London 1991). The interest in studying labdanes is heightened due to the wide range of biological activities of these compounds (Singh, M.; Pal, M.; Sharma, R. P. Planta Med., 1999, 65, 2-8.). They comprise a decalin system and a C-6 ring, which may be open or closed with an oxygen atom, as in manoyl oxide and its derivatives. Labdanes have been isolated from several plant families, such as Asteraceae, Labiateae, Cistaceae, Pinaceae, Cupressaceae, Taxodiaceae, Acanthaceae, Annonaceae, Caprifoliaceae, Solanaceae, Apocynaceae, Verbenaceae and Zingiberaceae. In addition they have been isolated from marine algae of the genus Laurence, from Taonia atomaria and from the red alga Chondria tenuissima.
The conifers are an important source of diterpenoids. Several labdanes have been detected in the neutral fraction of the oleoresin of Araucaria excelsa, including manool as well as nor-labdanes (Caputo, R.; Mangoni, L.; Monaco, P. Phytochemistry, 1972, 11, 839-840). A variety of biological activities have been associated with labdane diterpenes including antibacterial, antifungal, antiprotozoal, enzyme induction, anti-inflammatory modulation of immune cell functions, as well as cytotoxic and cytostatic effects against human leukemic cell lines. (K. Dimas et al. Planta Med. 1998, 208-211; K. Dimas et al. Leukemia Res. 1999, 217-234; K. Dimas et al. Anticancer Res. 1999, 4065-4072). In addition to the (antimicrobial, enzyme and endocrine related) properties mentioned above, it is interesting that many labdane type diterpenes also exhibit significant properties against cancer cells. A number of labdane type diterpenes tested exhibited remarkable antiproliferative and cytotoxic activities (Itokawa, H. et all. Planta Med. 1988, 311-315; K. Dimas et al. Planta Med. 1998, 208-211; K. Dimas et al. Leukemia Res. 1999, 217-234; K. Dimas et al. Anticancer Res. 1999, 4065-4072).
Labdane furanoids, and forscolin derivatives are the subject of several patents and applications, including European Patent Application 93103605.7; International Patent Publication No. WO 97/45099; International Patent Publication No. WO 91/02525; and International Patent Publication No. WO 85/03637.
Liposomes, or phospholipid vesicles, are self-assembled colloidal particles that occur naturally and can be prepared artificially (Lasic, D. D. Liposomes: from Physics to Applications. Elsevier), as shown by Bangham and his students in the mid-1960s (Bangham, A. D. ed. (1983) Liposomes Letters, Academic Press). At first, they were used to study biological membranes; several practical applications, most notably in drug delivery, emerged in the 1970. Today, they are a very useful model, reagent and tool in various scientific disciplines, including mathematics and theoretical physics, biophysics, chemistry, colloid science, biochemistry and biology. Liposomes were introduced as drug-delivery delivery vehicles in the 1970s. Early results were, however, rather disappointing, owing mainly to their colloidal and biological instability, and their inefficient and unstable encapsulation of drug molecules. Their utility was improved following basic research that increased our understanding of their stability and interaction characteristics.
In the scientific literature, there is reference to a great number of liposomic pharmaceutical forms. Many of these are in the clinical study stage and some other have been already registered and marketed. Among the medicines formulated in liposomic form, are econazole, amfotericin B, minoxidyl and some anticancer and antiviral medicines, which are in the clinical study stage.
It has been found that naturally occurring labdanes, such as labd-13-ene-8xcex1, 15-diol, labd-14-ene-8, 13-diol, and 3xcex2-hydroxy-labd-14-ene-8, 13-epoxy, exhibit biological properties in their pure state (Dimas et al., Planta Med. 1998) and may be useful as novel pharmaceutical and medicinal agents. The present invention deals with preparation of hydrated lipidic lamelar phases or liposomes particularly conventional and/or PEGylated and/or protein conjugated, containing the above compounds and their derivatives or plant extracts containing them, which are part of this invention. The compositions of the invention are useful for the treatment of neoplastic diseases.
As used herein the term xe2x80x9calkylxe2x80x9d refers to a straight or branched, saturated hydrocarbon containing from one to about twelve carbon atoms such as, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl, wherein one or more of the hydrogen atoms may be substituted.
As used herein the term xe2x80x9calkenylxe2x80x9d refers to a straight or branched hydrocarbon containing from one to about twelve carbon atoms where at least one carbon-carbon bond is unsaturated such as for example, vinyl, allyl and butenyl, wherein one or more of the hydrogen atoms may be substituted.
As used herein the term xe2x80x9calkynylxe2x80x9d refers to a straight or branched hydrocarbon containing from one to about twelve carbon atoms where at least one carbon-carbon bond is doubly unsaturated such as for example, acetylene, propynyl and butynyl, wherein one or more of the hydrogen atoms may be substituted.
As used herein the term xe2x80x9ccycloalkylxe2x80x9d refers to a cyclic hydrocarbon containing from three to about twelve carbon atoms such as, for example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, wherein one or more of the hydrogen atoms may be substituted.
As used herein the term xe2x80x9caralkylxe2x80x9d refers to a straight or branched, saturated hydrocarbon containing from one to about twelve carbon atoms, which is substituted with an aromatic ring such as, for example, benzyl and phenethyl, wherein one or more of the hydrogen atoms may be substituted.
As used herein the term xe2x80x9cheterocyclylxe2x80x9d refers to a cyclic hydrocarbon, wherein at least one carbon atom has been replaced by a heteroatom such as, for example, nitrogen, oxygen or sulfur, containing from three to about twelve atoms such as, for example, furan, pyran and imidazole.
As used herein the term xe2x80x9cdialkylaminoalkylxe2x80x9d refers to a straight or branched, saturated hydrocarbon containing from one to about twelve carbon atoms, which is connected to a tertiary amino group containing two alkyl groups such as, for example, diethylaminoethyl. Preferably, the dialklyaminoalkyl group is present as the acid addition salt resulting from reaction with either an inorganic or organic acid.
As used herein the terms xe2x80x9calkylthioketonesxe2x80x9d, xe2x80x9calkenylthioketonesxe2x80x9d, xe2x80x9calkynylthioketonesxe2x80x9d, xe2x80x9ccycloalkylthioketonesxe2x80x9d, xe2x80x9caralkylthioketonesxe2x80x9d and xe2x80x9cheterocyclothioketonesxe2x80x9d refer to a thioketone connected to a further radical.
As used herein the terms xe2x80x9calkylcarbonylxe2x80x9d, xe2x80x9calkenylcarbonylxe2x80x9d, xe2x80x9calkynylcarbonylxe2x80x9d, xe2x80x9ccycloalkylcarbonylxe2x80x9d and xe2x80x9caralkylcarbonylxe2x80x9d refer to a carbonyl connected to a further radical.
As used herein the term xe2x80x9csugarsxe2x80x9d refers hexoses or pentoses in their pyranose or furanose state or disaccharides containing hexose-hexose, pentose-pentose, hexose-pentose or pentose-hexose in their pyranose or furanose state. These sugars may be substituted with amino or halogen groups, preferably chlorine, bromine or iodine.
The labdanes of the present invention include:
A. Formula I,
LABD-13-ENE-8xcex1,15-DIOL (I) 
Wherein R wherein R is selected from the group consisting of H, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl, aralkylcarbonyl, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, dialkylaminoalkyl, alkylthioketones, alkenylthioketones, alkynylthioketones, cycloalkylthioketones, aralkylthioketones, heterocyclylthioketones and sugars.
B. Formula II
LABD-14-ENE-8, 13-DIOL (II) 
Wherein R is selected from the group consisting of H, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl, aralkylcarbonyl, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, dialkylaminoalkyl, alkylthioketones, alkenylthioketones, alkynylthioketones, cycloalkylthioketones, aralkylthioketones, heterocyclylthioketones and sugars.
C. Formula III
3xcex2-HYDROXY-LABD-14-ENE-8, 13-EPOXY 
Wherein R1 is xe2x95x90O, OR2 or a halogen selected from the group consisting of chlorine, bromine or iodine. R2 is selected from the group consisting of H, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl, aralkylcarbonyl, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, dialkylaminoalkyl, alkylthioketones, alkenylthioketones, alkynylthioketones, cycloalkylthioketones, aralkylthioketones, heterocyclylthioketones and sugars.
For the above derivatives, when R or R2 is dialkylaminoalkyl, the diethylaminoethyl group is preferred and a suitable acid addition salt is derived from inorganic or organic acid i.e hydrochloride, hydrobromide, sulfate, phosphate, acetate, oxalate, tartrate, citrate, maleate or fumarate. When R or R2 is aralkyl, phenylalkyl groups can be substituted by 1, 2 and 3 identical or different substituents such as halogen, C1-C3-alkyl, C1-C3-alkoxy, hydroxy, nitro, amino, trifluoromethyl, cyano and azodo.
In addition to the optical centers of the labdane nucleus, the substituents may also have chiral centers, which contribute to the optical properties of the compounds to the invention. This invention embraces all the optical isomers and racemic forms of the compounds according to the invention where such compounds have chiral centers in addition to those of the labdane nucleus.
Preferably, the labdanes used to prepare the compositions of the invention are isolated and/or purified labdanes. The labdanes of the invention may be at least 70% pure, 80% pure, 90% pure, 95% pure, 99% pure or 99.5% pure, as well as 100% pure. By xe2x80x9cpure,xe2x80x9d it is meant that the labdane is free from other compounds, thus, a 70% pure labdane preparation is one in which the labdane comprises 70%, by weight, of the total preparation.
The labdanes, labd-13-ene 8xcex1, 15-diol (I) and its derivative labd-13-ene 8xcex1, 15-yl acetate as well as 3xcex2-substituted -labd-14-ene-8, 13-epoxy when 3-substitue is hydroxy (OH) (III) or acetoxy (O Ac) groups have been detected into the extracts and essential oils of the plant Cistus creticus subsp. eriocephalus, and then identified for the first time (Anastassaki, Demetzos et al. Planta Med. 1999 735-739) using GC-MS (Gas Chromatography-Mass Spectrometry) methodology. The above compounds have been isolated in their pure state and their structures have been determined using spectroscopic methods, mainly NMR (Nuclear Magnetic Resonanse) (Demetzos et al. unpublished data). The labdane, labd-14-ene-8, 13-diol (II) namely sclareol has been isolated from Clary sage (Salvia sclarea Linn), as well as from Cistus incanus subsp. creticus (Ulubelen A., et al. Phytochemistry 1985, 1386; Demetzos C., Ph. D Thesis, Athens 1990).
The present invention provides liposomal formulations comprising one or more of the above described compounds. Any liposomal formulation known to those of skill in the art may be applied to the above described labdane compounds.
The lipids useful for the preparation of hydrated lipidic lamelar phases or liposomes comprising labdanes and/or their derivatives are described. The lipid molecules may be, but are not limited to, naturally occurring lipids such as HSPC (hydrogenated soy phosphatidylcholine), EPC (a mixture of saturated and unsaturated lipids from eggs) SPS (soy phosphatidylserine as sodium salt) and lipids isolated from natural sources (i.e. plants, marine organism and animal tissues) as mixtures of lipids and some synthetic lipids like: DSPC (distearoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine) and DPPC (dipalmytoylphosphatidylcholine) DOPC (dioleoylphosphatidylcholine), which are saturated esters of phosphatidylcholine. Polyethylene glycol (PEG)-lipid conjugates have been used to improve circulation times for liposomes encapsulated drugs and may be used in compositions of the present invention.
PEG-PE(phosphatidylethanolamine) have been used for preparing long circulating liposomes, and may be used in the compositions of the invention. PEG-lipid conjugates may also be used. Examples of PEG-lipid conjugates include 1,2-Diacy -sn-glycero-3-Phosphoethanolamine -N- [Methoxy(Polyethylene Glycol)-2000], in which the term acyl represents myristoyl , palmitpoyl, stearoyl and oleoyl groups.
Conventional or PEGylated liposomes containing cholesterol or cholic acid (transferosomes) in various concentrations by combining different phospholipids may also be utilized in the compositions of the invention. Cholesterol may regulate the stability of liposomes and therefore the inclusion of cholesterol in liposomes may be beneficial for the controlled release of the liposome associated compounds, such as the labdanes of the present invention. Because of the prolonged liposome circulation in blood and enhanced stability due to steric stabilization by surface-grafted polymers, the polymer-coated long-circulating liposomes have been referred to as sterically stabilized liposomes (Papahadjopoulos, D. et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88. 11460-11464). The optimal stability of this type of liposome is obtained at around 5 mol % of PEG-lipid (PEG molecular weight 2000 Da (Lasic, D. D. (1994) Angew. Chem, Int. Ed. Engl. 33, 1785-1799). Liposomes may be prepared by combining different synthetic lipids or natural lipids isolated from natural sources, such as lipids from plants and/or marine organisms and/or animal tissues. Liposomes may be prepared not only by combining different phospholipids but also by combining phospholipids with different levels of cholesterol and cholic acid (in its salt form).
Immunoliposomes are either conventional or sterically stabilized liposomes, which have specific proteins on their surface acting as recognition centers. Immunoliposomes may be prepared using the noncovalent biotin-advidin method and covalent bonding of proteins with the liposomes surface.
The PE derivatives of PEG with a terminal carbonyl group (Di acyl-PE-PEG-COOH) or with a terminal maleimidyl group (Di acyl-PE-PEG-Mal) may be synthesized according to K. Maruyama et al. B. B. A (1995) 1234, 74-80.
The use of immunoliposomes in the treatment of tumors resulted in a marked improvement in the drugs efficacy not only in comparison to the drug on its own but also compared to conventional liposomes.
Liposomes of different sizes and characteristics require different methods of preparation. The most simple and widely used method for preparation of MLV (Multilamelar Vesicles) is the thin-film hydration procedure in which a thin film of lipids is hydrated with an aqueous buffer at a temperature above the transition temperature of lipids. For lipophilic compounds such as the labdanes and their derivatives part of this invention, the REV (Reverse-Phase Evaporation), techniques is more suitable for the compounds encapsulation. In brief, different MLV liposomes composed of DSPC, DPPC, DMPC, DOPE, Soy Phosphatidylserine as sodium salt with or without cholesterol or cholic acid (as a salt) and PEGylated liposomes with or without cholesterol or cholic acid (as a salt) may be prepared by hydration with a buffer such as TES (N-tris-[hydroxymethyl] methyl 2-amino ethanesulfonic acid), MES (2-[N-morpholino] ethanesulfonic acid], HEPES (N-[2-hydroxyethyl]-piperazine-N-2-ethanesulfonic acid), after the removal of the organic solvent (Chloroform) in which labdanes and their derivatives have been dissolved.
The removal of the organic solvent in vacuum or under an inert gas results in the hydration of the lipids which form into multilayer liposomes upon vigorous shaking of the lipid film in an aqueous solution. The lipophilic labdanes incorporate into lipid bilayers, while the hydrophilic derivatives thereof are encapsulated in the liposomes. The aqueous medium used in hydrating the dried lipid film is preferably pyrogen free. The medium preferably contains physiological salt, such as NaCl, sufficient to produce a near-physiologic osmolarity (about 300 mOs).
The liposome dispersion is sized to achieve a size distribution of vesicles in a size range preferably between about 0.1 and 0.5 microns. The sizing serves to eliminate larger liposomes and to produce a defined size range having optimal pharmacokinetic properties. One preferred method for achieving the desired size distribution of liposome sizes is by extrusion of liposomes through a small-pore polycarbonate membrane sizes whose selected pore sizes such as 0.1, 0.2 or 0.4 microns, correspond approximately to the size distribution of liposomes after one or more passes through the membrane. Typically the liposomes are extruded through the membranes several times until the size distribution stabilises (Shokai et al, 1978). The liposomes dispersion is further treated to remove free labdanes, i.e. labdanes which are not intimately associated with the lipid bilayers. The suspension can be pelted by high-speed centrifugation after dilution, leaving free labdanes and very small liposomes in the supernatant. Another method uses gel filtration by molecular sieve chromatography to separate liposomes from free labdanes. Sephadex (G-75) gel filtration was used in order to remove the free labdanes.
In one embodiment, the final encapsulated liposomal labdane dispersion has the following characteristics:
1. Liposome sizes range between about 0.1 to 0.25 microns
2. Liposome-encapsulated labdanes about 80%-90%
3. The dispersion has a lipid concentration of at least 5 mg total lipid/ml, and near physiological osmolarity.
The dispersion may be sterilized by filtration through a conventional 0.22-micron depth filter.
The formulations of the invention are useful for treating mammalian cancers or conditions related thereto. By xe2x80x9ctreatingxe2x80x9d it is meant that the formulations are administered to inhibit or reduce the rate of cancer-cell proliferation in an effort to induce partial or total remission, for example, inhibiting cell division by promoting microtubule formation. For instance, the formulations of the invention are useful for treating, but not limited to, cancers of the blood, breast, lung, ovary, prostate, head, neck, brain, testes, kidney, pancreas, bone, spleen, liver, and bladder; AIDS-related cancers, such as Kaposi""s sarcoma; leukemia (e.g., acute leukemia such as acute lymphocytic leukemia and acute myelocytic leukemia); and the like. Preferably, the cancer to be treated is a leukemia. The formulations can be used alone or in combination with other chemotherapeutics. The dose of the composition of the invention to be administered, whether a single-unit dose, multi-unit dose, or a daily dose, will of course vary with the particular analog or derivative employed based on potency, administration route, patient weight, and the nature of the patient""s condition. The actual administered amount is to be decided by the supervising physician and may depend on multiple factors, such as, the age, condition, file history, etc., of the patient in question. By way of example, and not limitation, doses for parenteral use may be from 10-700 mg/kg, 10-500 mg/kg, 20-400 mg/kg, 50-300 mg/kg, 100-200 mg/kg. Doses for systemic use may be from 50-200 mg/m2, 75-150 mg/m2, or approximately 100 mg/m2.
The dose can be determined by a physician upon conducting routine experiments. Prior to administration to humans, the efficacy is preferably shown in animal models. Any animal model for cancer, preferably leukemia, known in the art can be used.
The subject, or patient, to be treated using the methods of the invention is an animal, e.g., a mammal, and is preferably human, and can be a fetus, child, or adult.
Preferably, the formulations of the invention are administered parenterally (intravenously, subcutaneously, intramuscularly, intraspinally, intraperitoneally, and the like). For parenteral administration, the formulations of the invention will normally be formulated as a solid, liquid, semisolid, gel, suspension, emulsion, or solution that, can be diluted in an aqueous medium to a concentration suitable for administration. The formulations of the invention can also be administered transdermally.
The present formulations can include additional pharmaceuticals and thus can serve as base formulation for polypharmacy. Such additional pharmaceuticals can be included and distributed in the formulation or added to the formulation prior to administration. For example, the formulations of the invention and other pharmaceuticals can be combined in an i.v. bag prior to administration. Additional pharmaceuticals can, for example, other chemotherapeutics.
The formulations of the invention can include additional suitable, pharmaceutically acceptable excipients. Preferred additional excipients, are those listed in the Physician""s Desk Reference, 54th edition, 881-887, Medical Economics Company (2000), i.e., water, aqueous vehicles such as saline, Ringer""s solution, or dextrose solution. Other examples of suitable excipients, such as binders and fillers are listed in Remington""s Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro, Mack Publishing Co. Easton, Pa., 1995 and Handbook of Pharmaceutical Excipients, 3rd Edition, ed. Arthur H. Kibbe, American Pharmaceutical Association, Washington D.C. 2000, both of which are incorporated herein by reference. Whatever excipient is incorporated into the present formulations, preferably, that excipient is sterile when added, or sterilized during the same process that sterilizes the formulation.
For parenteral administration as an aqueous solution, preferably, the present formulations are suitably buffered and isotonic. Furthermore, for parenteral administration, the formulations of the invention should be sterile. An embodiment of the present invention includes a sterilization step. The sterilization may be carried out in several ways, e.g., by using a bacteriological filter, by incorporating sterilizing agents into the composition, by irradiation, or by heating. Sterilization may be effected, for example, by filtration, e.g., through a 0.2 xcexcm pore size filter. Other methods of sterilizing known to those skilled in the art can also be employed. Suitable sterile and non-sterile excipients are commercially available from: EM Industries, Inc., Hawthorne, N.Y.; J. T Baker, Inc., Hayward, Calif.; Spectrum Quality Products, Inc., Gardena Calif.; Fisher Scientific International, Inc., Hampton N.H.; Aldrich Chemical Co., Inc., Milwaukee Wis.; Abbott Laboratories, Inc., North Chicago Ill.; Baxter Healthcare Corporation, Deerfield Ill.; and Amresco, Inc., Cleveland Ohio.
To formulate aqueous parenteral dosage forms for injection, an aqueous medium, e.g., physiological saline or purified water, paclitaxel solubilizers, and any additional components are mixed in sanitized equipment, filtered, and packaged according to well known methods in the art (for a discussion see e.g., Remington""s Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, Chapter 87). A formulation of the invention can by prepared in sterile form, such as a sterile solid, liquid, semisolid, gel, suspension, emulsion, or solution, preferably, as a sterile liquid concentrate that can be dissolved or dispersed in a sterile aqueous medium or any other injectable sterile medium prior to parenteral administration.
To formulate and administer transdermal dosage forms, well known transdermal delivery mediums such as lotions, creams, and ointments and transdermal delivery devices such as patches can be used (Ghosh, T. K.; Pfister, W. R.; Yum, S. I. Transdermal and Topical Drug Delivery Systems, Interpharm Press, Inc. p. 249-297, incorporated herein by reference). For example, a reservoir type patch design can comprise a backing film coated with an adhesive, and a reservoir compartment comprising a formulation of the invention, that is separated from the skin by a semipermeable membrane (e.g., U.S. Pat. No. 4,615,699, incorporated herein by reference). The adhesive coated backing layer extends around the reservoir""s boundaries to provide a concentric seal with the skin and hold the reservoir adjacent to the skin.
Gels, semisolids, and solid forms, containing the active can be prepared according to well known methods. For instance, by mixing in a standard V-blender, preferably, under anhydrous conditions. The homogeneous mixture can be passed through a screen mesh if desired. A comprehensive discussion on formulating solid forms is presented in Remington""s Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, Chapter 92, incorporated herein by reference.
The dosage form of the invention may be provided in single-unit dose container forms or multi-unit-dose container forms by aseptically filling suitable containers with the sterile solution to a prescribed active content as described above. It is intended that these filled containers will allow rapid dissolution of the composition upon reconstitution with appropriate sterile diluents in situ, giving an appropriate sterile solution of desired active concentration for administration. As used herein, the term xe2x80x9csuitable containersxe2x80x9d means a container capable of maintaining a sterile environment, such as a vial, capable of delivering a vacuum dried product hermetically sealed by a stopper means. Additionally, suitable containers implies appropriateness of size, considering the volume of solution to be held upon reconstitution of the vacuum dried composition; and appropriateness of container material, generally Type I glass. The stopper means employed, e.g., sterile rubber closures or an equivalent, should be understood to be that which provides the aforementioned seal, but which also allows entry for the purpose of introduction of diluent, e.g., sterile Water for Injection, USP, Normal Saline, USP, or 5% Dextrose in Water, USP, for the reconstitution of the desired active solution. These and other aspects of the suitability of containers for pharmaceutical products such as those of the invention are well known to those skilled in the practice of pharmaceutical arts.